RESUMO
Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes fibrocystin/polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope "knocked-in" to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location the in kidney, biliary duct, and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model.
Assuntos
Rim/metabolismo , Rim Policístico Autossômico Recessivo/genética , Domínios Proteicos/genética , Receptores de Superfície Celular/genética , Canais de Cátion TRPP/metabolismo , Animais , Cílios/metabolismo , Modelos Animais de Doenças , Epitopos/genética , Éxons/genética , Feminino , Imunofluorescência , Técnicas de Introdução de Genes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Fragmentos de Peptídeos/genética , Fenótipo , Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Polycystic kidney disease (PKD) is one of the most common life-threatening genetic diseases. Jared J. Grantham, M.D., has done more than any other individual to promote PKD research around the world. However, despite decades of investigation there is still no approved therapy for PKD in the United States. In May 2014, the University of Kansas Medical Center hosted a symposium in Kansas City honoring the occasion of Dr. Grantham's retirement and invited all the awardees of the Lillian Jean Kaplan International Prize for Advancement in the Understanding of Polycystic Kidney Disease to participate in a forward-thinking and interactive forum focused on future directions and innovations in PKD research. This article summarizes the contributions of the 12 Kaplan awardees and their vision for the future of PKD research.
Assuntos
Pesquisa Biomédica/tendências , Cílios/genética , Rim Policístico Autossômico Dominante/genética , Transdução de Sinais , Canais de Cátion TRPP/genética , Animais , Cílios/metabolismo , Genes Modificadores , Humanos , Túbulos Renais , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Terapia de Alvo Molecular , Complexos Multiproteicos/metabolismo , Fenótipo , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/patologia , Insuficiência Renal/prevenção & controle , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
Autosomal Dominant Polycystic Kidney Disease (ADPKD; MIM ID's 173900, 601313, 613095) leads to end-stage kidney disease, caused by mutations in PKD1 or PKD2. Inactivation of Pkd1 before or after P13 in mice results in distinct early- or late-onset disease. Using a mouse model of ADPKD carrying floxed Pkd1 alleles and an inducible Cre recombinase, we intensively analyzed the relationship between renal maturation and cyst formation by applying transcriptomics and metabolomics to follow disease progression in a large number of animals induced before P10. Weighted gene co-expression network analysis suggests that Pkd1-cystogenesis does not cause developmental arrest and occurs in the context of gene networks similar to those that regulate/maintain normal kidney morphology/function. Knowledge-based Ingenuity Pathway Analysis (IPA) software identifies HNF4α as a likely network node. These results are further supported by a meta-analysis of 1,114 published gene expression arrays in Pkd1 wild-type tissues. These analyses also predict that metabolic pathways are key elements in postnatal kidney maturation and early steps of cyst formation. Consistent with these findings, urinary metabolomic studies show that Pkd1 cystic mutants have a distinct profile of excreted metabolites, with pathway analysis suggesting altered activity in several metabolic pathways. To evaluate their role in disease, metabolic networks were perturbed by inactivating Hnf4α and Pkd1. The Pkd1/Hnf4α double mutants have significantly more cystic kidneys, thus indicating that metabolic pathways could play a role in Pkd1-cystogenesis.
Assuntos
Redes Reguladoras de Genes , Fator 4 Nuclear de Hepatócito , Rim Policístico Autossômico Dominante , Proteína Quinase C , Alelos , Animais , Proliferação de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure that is due to mutations in two genes, PKD1 and PKD2. Vascular complications, including aneurysms, are a well recognized feature of ADPKD, and a subgroup of families exhibits traits reminiscent of Marfan syndrome (MFS). MFS is caused by mutations in fibrillin-1 (FBN1), which encodes an extracellular matrix protein with homology to latent TGF-ß binding proteins. It was recently demonstrated that fibrillin-1 deficiency is associated with upregulation of TGF-ß signaling. We investigated the overlap between ADPKD and MFS by breeding mice with targeted mutations in Pkd1 and Fbn1. Double heterozygotes displayed an exacerbation of the typical Fbn1 heterozygous aortic phenotype. We show that the basis of this genetic interaction results from further upregulation of TGF-ß signaling caused by Pkd1 haploinsufficiency. In addition, we demonstrate that loss of PKD1 alone is sufficient to induce a heightened responsiveness to TGF-ß. Our data link the interaction of two important diseases to a fundamental signaling pathway.
Assuntos
Proteínas dos Microfilamentos/genética , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Fator de Crescimento Transformador beta/metabolismo , Doenças Vasculares/genética , Doenças Vasculares/metabolismo , Animais , Modelos Animais de Doenças , Epistasia Genética , Feminino , Fibrilina-1 , Fibrilinas , Estudos de Associação Genética , Haploinsuficiência , Heterozigoto , Humanos , Masculino , Síndrome de Marfan/etiologia , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Proteínas dos Microfilamentos/deficiência , Mutação , Rim Policístico Autossômico Dominante/complicações , Transdução de Sinais , Canais de Cátion TRPP/deficiência , Doenças Vasculares/etiologiaRESUMO
Autosomal-dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau (VHL) disease lead to large kidney cysts that share pathogenetic features. The polycystin-1 (PC1) and pVHL proteins may therefore participate in the same key signaling pathways. Jade-1 is a pro-apoptotic and growth suppressive ubiquitin ligase for beta-catenin and transcriptional coactivator associated with histone acetyltransferase activity that is stabilized by pVHL in a manner that correlates with risk of VHL renal disease. Thus, a relationship between Jade-1 and PC1 was sought. Full-length PC1 bound, stabilized and colocalized with Jade-1 and inhibited Jade-1 ubiquitination. In contrast, the cytoplasmic tail or the naturally occurring C-terminal fragment of PC1 (PC1-CTF) promoted Jade-1 ubiquitination and degradation, suggesting a dominant-negative mechanism. ADPKD-associated PC1 mutants failed to regulate Jade-1, indicating a potential disease link. Jade-1 ubiquitination was mediated by Siah-1, an E3 ligase that binds PC1. By controlling Jade-1 abundance, PC1 and the PC1-CTF differentially regulate Jade-1-mediated transcriptional activity. A key target of PC1, the cyclin-dependent kinase inhibitor p21, is also up-regulated by Jade-1. Through Jade-1, PC1 and PC1 cleaved forms may exert fine control of beta-catenin and canonical Wnt signaling, a critical pathway in cystic renal disease. Thus, Jade-1 is a transcription factor and ubiquitin ligase whose activity is regulated by PC1 in a manner that is physiologic and may correlate with disease. Jade-1 may be an important therapeutic target in renal cystogenesis.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Apoptose , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HEK293 , Meia-Vida , Proteínas de Homeodomínio/genética , Humanos , Rim/citologia , Rim/patologia , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Canais de Cátion TRPP/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/metabolismoRESUMO
The PKD1 gene is essential for a number of biological functions, and its loss-of-function causes autosomal dominant polycystic kidney disease (ADPKD). The gene is developmentally regulated and believed to play an essential role in renal development. Previous studies have shown that manipulating murine renal organ cultures with dominant-negative forms of the Pkd1 gene impaired ureteric bud (UB) branching. In the current study, we analyzed different stages of renal development in two distinct mouse models carrying either a null mutation or inactivation of the last two exons of Pkd1. Surprisingly, metanephric explants from Pkd1-deleted kidneys harvested at day E11.5 did not show defects of UB branching and elongation, estimated by cytokeratin staining on fixed tissues or by Hoxb7-GFP time-lapse imaging. However, renal explants from Pkd1-mutants isolated at day E14.5 showed impaired nephrogenesis. Notably, we observed cell migratory defects in the developing endothelial compartment. Previous studies had implicated the Pkd1 gene in controlling cell migration and collagen deposition through PI3 kinases. In line with these studies, our results show that wild-type explants treated with PI3-kinase inhibitors recapitulate the endothelial defects observed in Pkd1 mutants, whereas treatment with VEGF only partially rescued the defects. Our data are consistent with a role for the Pkd1 gene in the endothelium that may be required for proper nephrogenesis.
Assuntos
Glomérulos Renais/embriologia , Glomérulos Renais/fisiopatologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/fisiopatologia , Canais de Cátion TRPP/genética , Animais , Movimento Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Deleção de Genes , Glomérulos Renais/metabolismo , Camundongos , Mutação , Técnicas de Cultura de Órgãos , Inibidores de Fosfoinositídeo-3 Quinase , Canais de Cátion TRPP/metabolismoRESUMO
Mutations in PKD2 are responsible for approximately 15% of the autosomal dominant polycystic kidney disease cases. This gene encodes polycystin-2, a calcium-permeable cation channel whose C-terminal intracytosolic tail (PC2t) plays an important role in its interaction with a number of different proteins. In the present study, we have comprehensively evaluated the macromolecular assembly of PC2t homooligomer using a series of biophysical and biochemical analyses. Our studies, based on a new delimitation of PC2t, have revealed that it is capable of assembling as a homotetramer independently of any other portion of the molecule. Our data support this tetrameric arrangement in the presence and absence of calcium. Molecular dynamics simulations performed with a modified all-atoms structure-based model supported the PC2t tetrameric assembly, as well as how different populations are disposed in solution. The simulations demonstrated, indeed, that the best-scored structures are the ones compatible with a fourfold oligomeric state. These findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2.
Assuntos
Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Canais de Cátion TRPP/química , Sequência de Aminoácidos , Western Blotting , Cálcio/química , Cálcio/metabolismo , Dicroísmo Circular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Termodinâmica , Difração de Raios XRESUMO
Polycystic diseases affect approximately 1/1000 and are important causes of kidney failure. No therapies presently are in clinical practice that can prevent disease progression. Multiple mouse models have been produced for the genetic forms of the disease that most commonly affect humans. In this report, we review recent progress in the field and describe some of the outstanding challenges.
RESUMO
Autosomal dominant polycystic kidney disease is the most common inherited cause of end-stage kidney disease worldwide. Most cases result from mutation of either of 2 genes, PKD1 and PKD2, which encode proteins that form a probable receptor/channel complex. Studies suggest that a loss of function of the complex below an indeterminate threshold triggers cyst initiation, which ultimately results in dysregulation of multiple metabolic processes and downstream pathways and subsequent cyst growth. Noncell autonomous factors may also promote cyst growth. In this report, we focus primarily on the process of early cyst formation and factors that contribute to its variability with brief consideration of how new studies suggest this process may be reversible.
Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Humanos , Canais de Cátion TRPP/genética , Doenças Renais Policísticas/genética , Mutação , Rim Policístico Autossômico Dominante/genéticaRESUMO
PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.
Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Camundongos , Animais , Canais de Cátion TRPP/química , Rim Policístico Autossômico Dominante/genética , Modelos Animais de DoençasRESUMO
The polycystic kidney disease-1 (Pkd1) gene encodes a large transmembrane protein (polycystin-1, or PC-1) that is reported to function as a fluid flow sensor in the kidney. As a member of the transient receptor potential family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells. Here, we analyze two independent mouse models of PC-1, a knock-in (KI) mutant line and a hair cell-specific inducible Cre-mediated knock-out line. Both models exhibit normal MET channel function at neonatal ages despite hearing loss and ultrastructural abnormalities of sterecilia that remain properly polarized at adult ages. These findings demonstrate that PC-1 plays an essential role in stereocilia structure and maintenance but not directly in MET channel function or planar cell polarity. We also demonstrate that PC-1 is colocalized with F-actin in hair cell stereocilia in vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilli in vitro. These results not only demonstrate a novel role for PC-1 in the cochlea, but also suggest insight into the development of polycystic kidney disease.
Assuntos
Cílios/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Mecanotransdução Celular/fisiologia , Órgão Espiral/fisiologia , Canais de Cátion TRPP/fisiologia , Animais , Animais Recém-Nascidos , Cílios/genética , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Células Ciliadas Auditivas Internas/citologia , Células HeLa , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Humanos , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Canais de Cátion TRPP/deficiência , Canais de Cátion TRPP/genéticaRESUMO
Mutations of PKD1 and PKD2 account for most cases of autosomal dominant polycystic kidney disease (ADPKD). Compared with PKD2, patients with PKD1 typically have more severe renal disease. Here, we report a follow-up study of a unique multigeneration family with bilineal ADPKD (NFL10) in which a PKD1 disease haplotype and a PKD2 (L736X) mutation co-segregated with 18 and 14 affected individuals, respectively. In our updated genotype-phenotype analysis of the family, we found that PKD1-affected individuals had uniformly mild renal disease similar to the PKD2-affected individuals. By sequencing all the exons and splice junctions of PKD1, we identified two missense mutations (Y528C and R1942H) from a PKD1-affected individual. Although both variants were predicted to be damaging to the mutant protein, only Y528C co-segregated with all of the PKD1-affected individuals in NFL10. Studies in MDCK cells stably expressing wild-type and mutant forms of PKD found that cell lines expressing the Y528C variant formed cysts in culture and displayed increased rates of growth and apoptosis. Thus, Y528C functions as a hypomorphic PKD1 allele. These findings have important implications for pathogenic mechanisms and molecular diagnostics of ADPKD.
Assuntos
Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Animais , Apoptose/genética , Proliferação de Células , Células Cultivadas , Cistos/genética , Cães , Genótipo , Humanos , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Índice de Gravidade de DoençaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary disease affecting the kidneys, is caused in 85% of cases by mutations in the PKD1 gene. The protein encoded by this gene, polycystin-1, is a renal epithelial cell membrane mechanoreceptor, sensing morphogenetic cues in the extracellular environment, which regulate the tissue architecture and differentiation. However, how such mutations result in the formation of cysts is still unclear. We performed a precise characterization of mesenchymal differentiation using PAX2, WNT4 and WT1 as a marker, which revealed that impairment of the differentiation process preceded the development of cysts in Pkd1(-/-) mice. We performed an in vitro organ culture and found that progesterone and a derivative thereof facilitated mesenchymal differentiation, and partially prevented the formation of cysts in Pkd1(-/-) kidneys. An injection of progesterone or this derivative into the intraperitoneal space of pregnant females also improved the survival of Pkd1(-/-) embryos. Our findings suggest that compounds which enhance mesenchymal differentiation in the nephrogenesis might be useful for the therapeutic approach to prevent the formation of cysts in ADPKD patients.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cistos/prevenção & controle , Túbulos Renais/anormalidades , Mesoderma/efeitos dos fármacos , Rim Policístico Autossômico Dominante/prevenção & controle , Progesterona/administração & dosagem , Animais , Cistos/embriologia , Cistos/genética , Dilatação Patológica/embriologia , Dilatação Patológica/prevenção & controle , Feminino , Mesoderma/citologia , Camundongos , Camundongos Mutantes , Rim Policístico Autossômico Dominante/embriologia , Rim Policístico Autossômico Dominante/genética , Gravidez , Canais de Cátion TRPP/genéticaRESUMO
Polycystic kidney diseases (PKD) are inherited disorders characterized by fluid-filled cysts primarily in the kidneys. We previously reported differences between the expression of Cux1, p21, and p27 in the cpk and Pkd1 null mouse models of PKD. Embryonic lethality of Pkd1 null mice limits its study to early stages of kidney development. Therefore, we examined mice with a collecting duct specific deletion in the Pkd1 gene. Cux1 was ectopically expressed in the cyst lining epithelial cells of newborn, P7 and P15 Pkd1(CD) mice. Cux1 expression correlated with cell proliferation in early stages of cystogenesis, however, as the disease progressed, fewer cyst lining cells showed increased cell proliferation. Rather, Cux1 expression in late stage cystogenesis was associated with increased apoptosis. Taken together, our results suggest that increased Cux1 expression associated with apoptosis is a common feature of late stage cyst progression in both the cpk and Pkd1(CD) mouse models of PKD.
Assuntos
Apoptose/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Proteínas de Homeodomínio/genética , Doenças Renais Císticas/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Canais de Cátion TRPP/genética , Animais , Animais Recém-Nascidos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Progressão da Doença , Regulação para Baixo , Ativação Enzimática/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Canais de Cátion TRPP/metabolismo , Transfecção , Regulação para Cima/genéticaRESUMO
During murine kidney development, canonical WNT signaling is highly active in tubules until about embryonic days E16-E18. At this time, ß-catenin transcriptional activity is progressively restricted to the nephrogenic zone. The cilial protein genes PKD1 and PKD2 are known to be mutated in autosomal dominant polycystic kidney disease (ADPKD), and previous studies proposed that these mutations could lead to a failure to suppress canonical WNT signaling activity. Several in vitro studies have found a link between cilial signaling and ß-catenin regulation, suggesting that aberrant activity might contribute to the cystic phenotype. To study this, we crossed T-cell factor (TCF)/ß-catenin-lacZ reporter mice with mice having Pkd1 or Pkd2 mutations and found that there was no ß-galactosidase staining in cells lining the renal cysts. Thus, suppression of canonical WNT activity, defined by the TCF/ß-catenin-lacZ reporter, is normal in these two different models of polycystic kidney disease. Hence, excessive ß-catenin transcriptional activity may not contribute to cystogenesis in these models of ADPKD.
Assuntos
Cistos/etiologia , Rim Policístico Autossômico Dominante/metabolismo , Fatores de Transcrição TCF/genética , beta Catenina/metabolismo , Fatores Etários , Animais , Túbulos Renais/metabolismo , Camundongos , Camundongos Mutantes , Modelos Biológicos , Mutação , Rim Policístico Autossômico Dominante/genética , Fatores de Transcrição TCF/metabolismo , Canais de Cátion TRPP/genética , Transcrição Gênica , Proteínas Wnt/metabolismoRESUMO
Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.
Assuntos
Doenças Renais Policísticas/genética , Doenças Renais Policísticas/fisiopatologia , Sirolimo/farmacologia , Canais de Cátion TRPP/genética , Animais , Apoptose/fisiologia , Nitrogênio da Ureia Sanguínea , Divisão Celular/fisiologia , Modelos Animais de Doenças , Fibrose , Expressão Gênica/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Proteínas de Filamentos Intermediários/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais Coletores/patologia , Túbulos Renais Coletores/fisiopatologia , Túbulos Renais Distais/patologia , Túbulos Renais Distais/fisiopatologia , Camundongos , Mosaicismo , Proteínas do Tecido Nervoso/genética , Nestina , Fenótipo , Doenças Renais Policísticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR , Canais de Cátion TRPP/metabolismoRESUMO
Background: Multiple studies of tissue and cell samples from patients and preclinical models of autosomal dominant polycystic kidney disease report abnormal mitochondrial function and morphology and suggest metabolic reprogramming is an intrinsic feature of this disease. Peroxisomes interact with mitochondria physically and functionally, and congenital peroxisome biogenesis disorders can cause various phenotypes, including mitochondrial defects, metabolic abnormalities, and renal cysts. We hypothesized that a peroxisomal defect might contribute to the metabolic and mitochondrial impairments observed in autosomal dominant polycystic kidney disease. Methods: Using control and Pkd1-/- kidney epithelial cells, we investigated peroxisome abundance, biogenesis, and morphology by immunoblotting, immunofluorescence, and live cell imaging of peroxisome-related proteins and assayed peroxisomal specific ß-oxidation. We further analyzed fatty acid composition by mass spectrometry in kidneys of Pkd1fl/fl;Ksp-Cre mice. We also evaluated peroxisome lipid metabolism in published metabolomics datasets of Pkd1 mutant cells and kidneys. Lastly, we investigated if the C terminus or full-length polycystin-1 colocalize with peroxisome markers by imaging studies. Results: Peroxisome abundance, morphology, and peroxisome-related protein expression in Pkd1-/- cells were normal, suggesting preserved peroxisome biogenesis. Peroxisomal ß-oxidation was not impaired in Pkd1-/- cells, and there was no obvious accumulation of very-long-chain fatty acids in kidneys of mutant mice. Reanalysis of published datasets provide little evidence of peroxisomal abnormalities in independent sets of Pkd1 mutant cells and cystic kidneys, and provide further evidence of mitochondrial fatty acid oxidation defects. Imaging studies with either full-length polycystin-1 or its C terminus, a fragment previously shown to go to the mitochondria, showed minimal colocalization with peroxisome markers restricted to putative mitochondrion-peroxisome contact sites. Conclusions: Our studies showed that loss of Pkd1 does not disrupt peroxisome biogenesis nor peroxisome-dependent fatty acid metabolism.
Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Proteína Quinase C/metabolismo , Animais , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Mutação , Peroxissomos/metabolismo , Doenças Renais Policísticas/genética , Rim Policístico Autossômico Dominante/genéticaRESUMO
Polycystin-1 (PC1) has an essential function in renal tubular morphogenesis and disruption of its function causes cystogenesis in human autosomal dominant polycystic kidney disease. We have previously shown that recombinant human PC1 is cis-autoproteolytically cleaved at the G protein-coupled receptor proteolytic site domain. To investigate the role of cleavage in vivo, we generated by gene targeting a Pkd1 knockin mouse (Pkd1(V/V)) that expresses noncleavable PC1. The Pkd1(V/V) mice show a hypomorphic phenotype, characterized by a delayed onset and distal nephron segment involvement of cystogenesis at postnatal maturation stage. We show that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist. Our study establishes a critical but restricted role of cleavage for PC1 function and suggests a differential function of the two types of PC1 molecules in vivo.